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University of Santo Tomas Biochemistry

Faculty of Medicine and Surgery John Mark Villena


Class of 2017 Section D

Laboratory Reviewer 2
 
Experiment 3: Factors That Affect Enzyme Activity
Enzymes
• conjugated proteins that catalyze biological reactions by lowering the energy of activation (ΔG)
• act on specific substrates & are regenerated after every reaction

• Holoenzyme – catalytically active form of an enzyme


o Apoenzyme (Inactive, protein portion) + Cofactor (Activator, Non-protein portion)
o Cofactors
§ Organic
• Tightly Bound: Coenzyme
• Loosely Bound: Cosubstrate
§ Inorganic Metal Ions
• Tightly Bound: Metalloenzyme
• Loosely Bound: Activators

Alkaline Phosphatase
• Enzyme used in the experiment extracted from rat intestines
• Class III: Hydrolase à non-specific metalloenzyme
• Activator: Magnesium Ions - from MgCl 2

• Optimum pH
o 9.80 – 10.00
o maintained by Glycine Buffer (Buffering range: 8.80 – 10.60)
• Optimum Temperature
o 37ºC
o maintained by water bath
• Substrate: Sodium B-Glycerophosphate
• Reaction Catalyzed:

• Trichloroacetic Acid (TCA)


o After incubation of enzyme with substrate, we must ensure that the reaction is no longer proceeding to be
able to measure the product accurately. We do this by precipitating the enzyme to make sure that no more
product is formed during measurement.
• Enzyme activity is measured by how much phosphate is liberated
o Phosphate + Molybdate II à Phosphomolybdate
o Phosphomolybdate + Amino Naphthol Sulfonic Acid (Reducing agent) à Molybdenum Blue
o Molybdenum blue is a blue complex which absorbs at 660nm

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
• Dialysis of Alkaline Phosphatase
o Dialysis
§ separation of the naturally-bound Magnesium Ions from
the enzyme structure
§ uses a selectively permeable membrane (dialyzing bag)
with pores to allow the Small Magnesium ions to go out
of the dialyzing bag & allow the apoenzyme/inactive
enzyme to be trapped within the bag
§ must be done in a cold temperature solution for the
enzyme to be inactive during the dialysis
o Enzymes to be used per experiment
§ Undialyzed/Active Holoenzyme
• Effect of pH & Temperature
• To ensure a catalytically active enzyme that would ONLY be affected by the variables of
temperature and pH
§ Dialyzed/Inactive Apoenzyme
• Effect of Activators & Inhibitors
• The variable being tested in the effects of Activators and Inhibitors was the presence of
cofactors which would affect enzyme activity. We would not be able to observe the effect
of its absence/pressence if there are naturally-bound cofactors to the enzyme
• Dialysis would eliminate this problem by removing the naturally-bound cofactors to be able
to observe the true effects of activators & inhibitors

• Factors Affecting Activity

o Enzyme Concentration (Direct linear)


§ éEnzyme Concentration: éActive sites present to catalyze reaction:
éProducts formed: éActivity
§ Constants
• Substrate Concentration is constantly high
• Optimum enzyme environment: pH & Temperature
• Enzyme activators are present
§ Saturation of active sites is impossible as more enzyme is added over time
§ Activity only decreases when all the substrate is consumed

o Substrate Concentration (Hyperbolic curve)


§ éSubstrate Concentration: éProducts formed: éActivity until
• Point of Saturation
o Enzyme activity plateus: further increase in substrate
concentration no longer increases enzyme activity
o All the active sites are already saturated/occupied
o Maximum activity

o Temperature (Bell-shaped curve)


§ Ascending Limb
• éTemperature: éKinetic Energy: éCollision frequency of reacting
molecules: éProximity & Orientation catalysis: éActivity
§ Optimum Temperature
• temperature at which enzyme is at its maximum activity
§ Descending Limb
• éTemperature: Denaturation of enzyme – unfolding of the
polypeptide chain and loss of catalytic activity
§ Temperatures tested: 5ºC, 25ºC, 37ºC, 60ºC

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

o pH (Bell-shaped curve)
§ Ascending Limb
• épH: éActivity
• during this time, we modify the amino acid charged groups toward
the proper functional enzyme folding pattern
§ Optimum pH
• pH at which enzyme is at its maximum activity
• optimum/proper/functional folding pattern of the enzyme
§ Descending Limb
• épH: Denaturation of enzyme – unfolding of the polypeptide chain
and loss of catalytic activity
§ pH Tested: 1.00, 7.00, 8.50, 10.50
§ pH was measured by pH meter
§ pH was altered by HCl (acids decrease pH) & NaOH (bases increase pH)

o Activators
§ Metal ion cofactors act as activators of catalysis by
• binding to substrate to orient them properly for reaction
• mediating oxidation-reduction reactions through reversible changes in the metal ions
oxidation state
• electrostatically stabilizing or shielding negative charges
§ Activators Tested: Mg+2 (from MgCl ), Ca+2 (from CaCl ), Fe+3 (from FeCl )
2 2 3

§ Magnesium Ions
• Known activator of Alkaline Phosphatase
• It localizes the pi electrons present in the double bond of PO4- to facilitate stabilization of
the negative charge.
• Stabilization/masking of the charge will allow the enzyme’s active site to nucleophillically
attack the substrate making catalysis possible

o Inhibitors
§ chemicals that bind to the enzyme and reduce the rate of enzymatic
reactions
§ inhibitors tested: chelating agents – bind to metal cofactors & make them
unavailable to the enzyme, hence inactivating it.
• Ethylene Diaminetetraacetic Acid
o better inhibitor as it has 4 COO- which could chelate 2Mg 2+

• Citrate
o has only 3 COO- which could chelate only 1Mg 2+

 
 
 
 
 
 
 
 
 
 

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

Experiment 4: Kinetics of Enzyme Reaction


Enzyme Kinetics
• quantitative measurement of the rates of enzyme-catalyzed reactions (how fast/how slow a reaction occurs)
• systematic study of factors that affect these rates (what makes it fast/slow)
• Measurement of enzyme activity
o Amount of Products produced per unit time (rate/velocity of appearance of products) – most common
o Amount of Reactants consumed per unit time (rate/velocity of disappearance of reactants)
In the Active site..

Where: S = substrate
E = enzyme
ES = enzyme-substrate complex
K , K and K = rate constants
1 =1 2

• Enzyme reversibly combines with its substrate to form an ES


complex
• K1 is the rate at which ES is formed, K-1 is the rate at which it is degraded back to E+S
• Formation of the ES Complex leads to the formation of a transition state species
• TS species is unstable, as it stabilizes it breaks down into the product, regenerating the free enzyme.
• K2 is the catalytic constant, it is the rate at which the product is formed. Slowest/rate-limiting step.

Michaelis-Menten Kinetics
• Describes how reaction velocity varies with substrate
concentration
• Graph
o V = Initial velocity
o Vmax = Maximum velocity, 100% of enzyme active
site is saturated
o ½ Vmax = 50% of enzyme active site is saturated
with substrate
o Km
§ Michaelis Constant (K-1+K2)/K1
§ (x =Km, y=1/2Vmax)
• get half the Vmax then trace to
the x-axis, that is your Km
§ reflects the affinity of the E for the S
• Small Km = High Affinity
o low [S] is needed to half-
saturate the enzyme
• Big Km = Low Affinity
o high [S] is needed to half-saturate the enzyme
o [S] = Substrate concentration
• Assumptions
o Assumption of Equilibrium
§ S binds reversibly with E
• initially P is small: E+SàES
• when P accumulates, reaction reverses ESàE+S
• Until equilibrium is reached catalyzed by the
enzyme
o Assumption of Briggs & Haldane
§ steady state assumption
§ rate of synthesis (E + S --> ES) = rate of degradation (ES --> E + P)
o Substrate Concentration is far > Enzyme concentration
§ enzyme will be limiting when the active sites are occupied
o The rate of the reaction is measured the moment S + E comes in contact: Initial Velocity
§ at the Initial Portion of the reaction
§ A lot of substrates available for conversion & no products are formed yet that may inhibit the
reaction (via negative feedback) so we are able to obtain maximum velocity at the start of the
reaction

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
• Reaction
o Pseudo-1 order
st

§ linear part of the graph where [S]<Km


§ reaction velocity is directly proportional to substrate concentration
o Zero order
§ High [S] > Km
§ flat portion of the graph
§ rate of the reaction is constant/not changing, that is called the Vmax
§ rate of reaction is independent of substrate concentration

Lineweaver-Burke Plot
• graphical linearization of the Michaelis-Menten Kinetics to be able to determine Km and Vmax
• double reciprocal plot

Constructing the Graphs


• Get the Absorbance readings of the 4 flasks
• Compute for the substrate concentration per flask using C1V1=C2V2
• Use a standard curve to convert Absorbance à PO4 liberated
• Create 1 graph per flask
o X-axis: Time in minutes
o Y-axis: PO4 liberated
• Obtain the Vi per graph
o y = mx + b
o Initial velocity = slope (m) = Products Liberated/Change in time
• Construct the Michaelis Menten Graph
o X-Axis: Substrate Concentration
o Y-Axis: Initial Velocity
• Construct the Lineweaver-Burke Plot
o X-Axis: 1/Substrate Concentration
o Y-Axis: 1/Initial Velocity
• Obtain Km and Vmax
o X-intercept = -1/Km
o Y-intercept = 1/Vmax

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

Experiment 5: Tests to Diagnose Diabetes Mellitus


Glucose
• Aldohexose serving as the primary carbohydrate source for energy utilized by the body’s metabolic pathways
• Hormonal Responses to Blood Glucose Levels
o Glucagon – released during hypoglycemia to increase BGL
o Insulin – released during hyperglycemia to decrease BGL
Diabetes Mellitus
• Group of metabolic diseases characterized by high blood glucose
levels (hyperglycemia) as its entry into the cells is impaired
• Types of DM
o Type I (Insulin dependent)
§ defect in pancreatic B-cell secretion of insulin during
hyperglycemia
§ defective Insulin produced
o Type II (Insulin independent)
§ B-cell failure
• amount of insulin produced is inadequate to
stop Gluconeogenesis in the liver
§ Insulin resistance
• Diagnosis
o Fasting Plasma Glucose (FPG)
§ measures the fasting level of blood glucose to screen for diabetes or prediabetes
§ sets the baseline glucose level
§ Requirement for Overnight Fast
• No caloric Intake
• Water only
• 8hours
§ Lower sensitivity than OGTT

o Oral Glucose Tolerance Test (OGTT)


§ Tests the down regulating capacity of the human body in response to hyperglycemia
• Time Zero: Determine baseline glucose level using FPG
• Ingest oral dose of glucose
o Adults: 75g (25g/100mL: 75g/300mL)
o Children: 1.75g/kgBW, 75g MAX
o Consume in 5min or less
• Time 2hour: Determine glucose level 2-hours after ingestion
§ Detects mild/moderate diabetes
§ Requirements
• Stable diet
o 100-150g/day Carbohydrate for 3 days prior to the test
o Inadequate Carbohydrate may decrease glucose tolerance, may be interpreted as
diabetic
• Stable: Weight, Level of exercise
• No acute illness and/or recent hospitalization
• Fast for 8-12 hours overnight: No caloric intake, medication, smoking. Just water
o Hormonal diurnal effect on glucose
o Preferrably in the AM - glucose levels are higher in the morning than in the
afternoon
§ Glucose Oxidase Test

Oxidation  

Red  Brown  
Semiquinone  
*Read  absorbance  

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
o Glycosylated Hemoglobin A1c (HbA1c)
§ excess blood glucose is non-enzymatically bound to amino acids of certain proteins like Hemoglobin
of RBC; more glucose in the blood stream, more Glycosylation of proteins would occur
§ measures the amount of Glucose (in %) that is attached to Hemoglobin of RBC
§ Since RBCs live for 120 days, Hb1Ac test shows the average blood sugar for the past several
months & not just the moment of post-fasting hence, it is used to monitor patient compliance to
DM treatments
§ Not affected by sudden fluctuations in blood glucose
§ Clinical Interpretations
• 6% 120 mg/dl (Normal)
• 8% 180 mg/dl (not too bad)
• 10% 240 mg/dl (not good)
• 13% 330 mg/dl (dangerous)
§ False Elevations
• Restricting carbohydrate intake may alter baseline results
• Illness may conceal a person’s actual glucose metabolism when healthy
• Exaggerated amounts of glucose may lead to erroneous results
GLUCOSE TOLERANCE CURVE
• Normal Patient
o Peak – 1 hour st

o After which levels return to Fasting


o Levels
§ Fasting = 60-100 mg/dl
§ 2hr PG < 140 mg/dl
• Diabetic Patient
o Peak – 2 hour nd

o After which levels decrease


o Levels
§ Fasting >/= 120 mg/dl
§ 2hr PG >/= 200 mg/dl

*notice the massive difference in the glucose levels

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

Experiment 6: Establishing A Lipid Profile


• All plasma lipids are present as lipoprotein complexes
o Triglyceride
o Total Cholesterol
o HDL & LDL Cholesterol
• Specimen: Blood Plasma
o Fasted 12-14hours before the test
o No caloric intake: Only water
o No alcohol for 24hours before the test
• Determination of lipid profile & presence of risk factors is useful in screening for
o genetic/acquired predisposition to coronary heart disease
o monitoring of a course of treatment for hyperlipoproteinemia

• Determination of Triglycerides
o Nature
§ TAGs/Neural Fats exists in the plasma
§ Formed by esterification of glycerol + 3 fatty acids
§ Major fat in the diet (>90% of dietary fat)
§ Main storage of fats in man; 95% of adipose tissue lipids
§ Found in plasma as part of lipoproteins in various concentration
§ Highest during absorption after meals (peak at about 4 to 6 hours after a meal, exogenous lipids
should be cleared from plasma before analysis)

o Assay
§ Hydrolysis of TAG to Glycerol + 3 Free Fatty Acids by Lipase
§ Glycerol is phosphorylated by Glycerol Kinase & ATP forming Glycerol-3-Phosphate
§ Glycerol-3-Phosphate is oxidized by Glycerol Phosphate Oxidase to Dihydroacetone Phosphate +
Hydrogen Peroxide (H O )2 2

§ (H O ) oxidizes 4-aminoantipyrene to Quinoneimine via Peroxidase


2 2

§ Quinoneimine is a red chromophore which is measure spectrophotometrically


§

o Specimen: Serum
o Reagents
§ PIPES buffer (pH 7.5), 4-chlorophenol, 4-aminoantipyrine, Magnesium ions, ATP, Lipases, Peroxidase,
Glycerol kinase, Glycerol-3-phosphate oxidase
o Computation
§ [TAG]mg/dL = (ASX/ASTD) x 200
§ [TAG]mmol/L = (ASX/ASTD) x 2.28
o Clinical Interpretation
§ SUSPECT IF ABOVE: 150mg/dL (1.71mmol/L)
§ ELEVATED RISK IF ABOVE: 200mg/dL (2.28mmol/L)

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

• Determination of Total Cholesterol


o Nature
§ A total cholesterol test measures all the cholesterol in your blood.
§ Cholesterol is a soft, wax-like substance found in all parts of the body.
§ too much cholesterol can clog your arteries and lead to heart disease.
§ This test is often done to determine your risk for coronary artery disease.
§ High blood cholesterol and triglycerides have been linked to heart attack and stroke.

o Assay: 500nm

o Specimen: Serum
o Computation
§ [Cholesterol]mg/dL = A x 553
§ [Cholesterol]mmol/L = A x 14.3
o Clinical Interptretation: Hypercholesterolnemia
§ SUSPECT IF ABOVE: 220mg/dL (5.7mmol/L)
§ ELEVATED RISK IF ABOVE: 260mg/dL (6.7mmol/L)

• Determination of HDL Cholesterol


o Nature
§ high-density lipoprotein/ "good" cholesterol
§ The main function of HDL is to help soak up excess cholesterol from the walls of blood vessels and
carry it to the liver, where it breaks down and is removed from the body in the bile.
§ The laboratory test for HDL actually measures how much cholesterol is in each high-density
lipoprotein particle
o Assay
§ Precipitation of Chylomicron, VLDL & LDL by Phosphotungstic Acid + Magnesium Ions
§ Centrifuge to leave HDL in the supernatant fluid
§ Cholesterol Determination
• Cholesterol Liquicolor Reagent
• Same as reaction in determining Total Cholesterol
o Computation
§ [HDL-C]mg/dL = A x 274
§ [HDL-C]mmol/L = A x 7.09

• Determination of LDL Cholesterol


o Nature
§ low-density lipoprotein/ "bad" cholesterol
§ Lipoproteins are made of fat and protein. They carry cholesterol, triglycerides, and other fats, called
lipids, in the blood to various parts of the body.
§ The LDL test is usually done as part of a lipid panel, which also checks total cholesterol, HDL,
and triglyceride levels.
§ LDL carries cholesterol to various tissues throughout the body.
§ Too much LDL is linked to cardiovascular disease.
o Assay
§ Computed from Total Cholesterol Concentration
• [LDL-C]mg/dL = (Total Cholesterol) – [(TAG/5) + HDL-C]
• [LDL-C]mmol/L = (Total Cholesterol) – [(TAG/2.2) + HDL-C]
o Clinical Interpretation
§ Suspicious: 150mg/dL (3.9mmol/L)
§ Elevated Risk: 190mg/dL (4.9mmol/L)

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

Sources of Cholesterol
• Dietary absorption – exogenous cholesterol
• De Novo Biosynthesis – endogenous cholesterol

A. Entry of Cholesterol in the body thru the diet


• Stomach: Dietary Fat à Small Intestines: Fat Emulsification by Bile Salts = TAGs
• Pancreatic secretions – catalyzes the hydrolysis of TAG à 2(Free Fatty Acids or) + 1 (2-Monoacylglycerol)
o Pancreatic Lipase – hydrolyzes positions 1 & 3 of the glycerol moiety
o Colipase – cause pancreatic lipase to be more active
o Bicarbonate – raises pH to optimal level
o Esterases – remove fatty acids from compounds
o Phopholipases – diigest phospholipids
• The 2 FFAs & 1 2-MG are then packaged into micelles
• Micelles then travel through unstirred layer of water to the microvilli where FFAs and MGs are absorbed by Intestinal
epithelial cells
• Within intestinal epithelial cells, FFAs and MGs recombine to form TAGs in the SER
• TAGs are then packaged into chylomicrons which are then secreted by intestinal epithelial cells into the chyle of the
lymphatic system and enter the blood via the thoracic duct
• Chylomicrons enter the blood within 1-12 hours after a meal
• Lingual and Gastric lipases hydrolyze short and medium chain fatty acids and don’t need to be packaged into
chylomicrons, they enter the blood directly
B. De Novo Cholesterol Synthesis
• Biosynthesis of new/endogenous cholesterol
• Occurs in the Cytoplasm & Endoplasmic Reticulum of liver (major) & intestines, adrenal glands, ovaries, skin (minor)
• All carbons of Cholesterol comes from Acetyl-CoA
• Rate Limiting/Committed step: HMG-CoA Reductase

• Regulation
o Regulation of HMG-CoA Reductase
§ Competitive inhibition - statin drugs: êActivity
§ Feedback control - éDietary cholesterol, éBile acids: êActivity
§ Fasting - êActivity due to unavailability of Acetyl-CoA
§ Hormonal/Covalent modification – Phosphorylation/Dephosphorylation
• Glucagon - Activates Protein Kinase (adds PO4): êActivity
• Insulin – Activates Protein Phosphatase (removes PO4): éActivity
§ Transcriptional control – rate of synthesis of HMGCoA reductase is controlled by sterol regulatory
binding protein (SREBP) when cholesterol levels drop
o Regulation of the LDL Receptor
§ éIntracellular cholesterol: êLDL Receptor synthesis: No more cholesterol uptake
o Regulation of rate of cholesterol esterification and removal of free cholesterol
§ Cholesterol à Bile Acids
§ Cholesterol à Cholesteryl Esters via ACAT

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

Hyperlipidemia

Type Plasma Lipid Appearance of Principle Treatment


Lipoprotein
Cholesterol Triglycerides Plasma Upon Abnormality
Standing Increased
I
Abdominal spasm,
anorexia, Creamy layer on Chylomicrons Diet Control
hepatosplenomegaly,
Normal/é é
top, clear below
lipidemiaretinalis, eruptive
xanthomas

II-A
Accelerated
atherosclerosis, premature Normal Clear B-Lipoprotein Statin drugs
coronary artery disease, é
Niacin
corneal arcus, Cholestyramine
tendinousxanthomas on
achilles tendon, tuberous
xanthoma

II-B Fibrate
*same as II-A é é Clear/Slightly Pre-B Statin drugs
Turbid B-Lipoprotein Niacin

III
Palmarxanthoma on hands
and fingertips, Turbid, creamy Broad B Band Fibrate
tuboeruptivexanthoma over é é
layer sometimes Statin drugs
elbow and knees,
premature atherosclerosis

IV Fibrate
Diabetes, hypertension, Normal/é é Clear/Turbid Pre-B Statin drugs
obesity, atherosclerosis Niacin

V
Abdominal pain, eruptive
xanthoma on extensor Creamy layer on Chylomicrons Niacin
surfaces of arms and legs,
Normal/é é
top, turbid below Pre-B Fibrate
hepatosplenomegaly,
lipidemiaretinalis,
pancreatitis, peripheral
neuropathy

Treatment MOA
• Statins – decreases blood cholesterol levels by competitively inhibiting HMG-CoA Reductase
thus inhibiting cholesterol biosynthesis
• Niacin – decreases VLDL & stimulates reverse cholesterol transport by HDL
• Cholestyramine – sequesters bile acids & excretes them to facilitate bile acid synthesis
from cholesterol to decrease cholesterol levels
• Fibrates – reduces VLDL production & increasing TAG clearance from the blood
• Ezetimibe – inhibits GI cholesterol absorption by ATP-binding cassette transporter inhibition

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

Experiment 7A: Determination of Urea & BUN


Urea (Carbamide)
• principal excretory product of protein metabolism
• constitutes nearly 50% of NPN (Non-protein nitrogenous) compounds in the blood
• Metabolism
o LIVER: synthesis in the liver mitochondrion + cytosol via the Krebs-Henseleit Cycle (Ornithine/Urea
Cycle)
o BLOOD PLASMA: transport from liver à kidneys
o KIDNEY: filtered by glomerulus & excreted in the urine (30g Urea/day)

Blood Urea Nitrogen (BUN)


• amount of Nitrogen in the blood that comes from Urea
• varies directly with protein intake & inversely with rate of Urea excretion
o éProtein Intake: éBUN
o éUrea Excretion: êBUN
• Factors affecting Level of BUN in plasma
o Renal Function - MORE EXCRETION = LESS BUN
o Dietary Protein Intake – MORE DIETARY PROTEIN = MORE BUN
o Liver Function – RATE OF PROTEIN BREAKDOWN = RATE OF UREA SYNTHESIS
• Modified Berthelot Reaction
o Assay to measure BUN
o Urea can be measured from urine & blood plasma

Urease Urease hydrolyzes


Ammonia Urea to Ammonia &
*Sample is in the form of Carbamic Acid
blood plasma as Urea is Carbamic
Urea Acid
transported via the blood
Spontaneously Carbamic Acid
  Ammonia spontaneously
CO2 decomposes to
Ammonia & CO 2
Carbamic
Acid

2 Ammonia + CO2 SUMMARY:


1 Urea = 2 Ammonia + CO 2

Ammonia then reacts with Salycilate, Sodium


Nitroprusside & Alkaline Hypochlorite (NaOH +
Sodium Hypochorite) to form 2-2 Dicarboxy
Indophenol (Blue Green Chromophore which
absorbs light at 578nm
Intensity of Blue Green Color = Concentration of 2-2 Dicarboxy Indophenol = Concentration of Urea

REACTION SUMMARY:
 
Nitroprusside
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
o Reagents
§ RGT1: BUN Enzyme Reagents
• Phosphate Buffer (pH 7.00) – to maintain optimum pH of Urease
• Sodium Salicylate – condenses with Ammonia to form colored compound
• Sodium Nitroprusside – catalyzes formation of colored compound
• EDTA – chelates metal ions
§ RGT2: Color Developing Reagent
• Phosphate Buffer (pH < 13.00) – maintains constant alkaline condition for color
dev’t
• Sodium Hydroxide – base, donates OH ions for an alkaline environment
• Sodium Hypochlorite – condenses with Ammonia to impart color
§ ENZ: Enzyme Concentrate
• Urease – catalyzes the hydrolysis of Urea to Ammonia & CO2
§ STD: Standard Reagents – for us to be able to have standard readings for sample
computation
• Urea: 80mg/dL (13.3mmol/L)
• BUN Equivalent: 37.28mg/dL (6.2mmol/L)
• Sodium Azide: 0.0095%
o Normal Value
§ Urea = 10 – 50 mg/dL (1.7 - 8.3 mmol/L)
§ BUN = 4.66 – 23 mg/dL (0.79 – 3.86 mmol/L)
o Computation
§ Absorbance of the sample / Absorbance of the Standard (A /A ) SX STD

§ (A /A ) x factor
SX STD

§
We can get both Urea concentration & BUN concentration from the same sample
§
We can also interconvert from Urea concentration à BUN concentration and vice versa
• [BUN] = 0.466 x [Urea]
• [Urea] = 2.14 x [BUN]
• Indications of BUN Measurement
o éBUN (Uremia)
§ Azotemia (Kidney Malfunction): Chronic nephritis, Glomerulonephritis, Polycystic kidney
§ Urinary Tract obstruction – increase back diffusion of Urea to renal tubules
§ Heart Failure
§ Dehydration -
§ High-Protein Diet
§ Muscle wasting – during starvation, body proteins will be broken down & [Urea] will increase
o êBUN
§ Liver Damage/Disease – Urea Cycle impairment: Urea synthesis is impaired: No BUN
§ Malnutrition/Starvation: Low Protein Intake – No protein to catabolize: No BUN
§ 2 /3 Trimester of Pregnancy
nd rd

§ Overhydration
§ Alcoholism
o Variations
§ Adult > Children – growing children use amino acids for synthesis, not breakdown: growth
§ Male Adult > Female Adult – higher protein catabolism in male vs female
• BUN to Creatinine Ratio
o differential diagnosis
§ Creatinine in blood also tells how well the kidneys are working
§ High Creatinine = Kidney Malfunction
o éBUN, Normal Creatinine
§ relative decrease of blood flow to the kidney (as seen in heart failure or dehydration)
without indicating any true injury to the kidney.
o éBUN, éCreatinine
§ Kidney malfunction
• Drugs that interfere with BUN measurement
o False Increase: Allopurinol, Aminoglycosides, Amphotericin B, Carbamazepine, Cephalosporins, Cisplatin, Colistin, High-
dose aspirin, Indomethacin, Methotrexate, Methyldopa, Penicillamine, Probenecid, Propranolol, Rifampin, Diuretics
o False Decrease: Chloramphenicol, Streptomycin

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

Experiment 7B: Determination Serum Transaminases


Transaminase/Aminotransferase
• Catalyzes the transfer of an amino group between Amino Acid & Alpha-Keto Acid
• Pyridoxal Phosphate (PLP) as the coenzyme

• Transamination reactions are important in metabolism because of their function in the synthesis & degradation of
amino acids
• AA -> a-KA -> TCA -> Energy source/Gluconeogenesis
• Compartmentalized intracellularly in specific organs
o Pressence in the Blood means
§ cellular destruction – Hepatocellular Disorders, Skeletal muscle disorders
§ tissue infarction – Myocardial infarction
§ necrosis
• Specimen: Serum, Heparinized Plasma or EDTA Plasma. Avoid Hemolysis
• Transaminases to be tested

o Aspartate Transaminase (AST)


§ Catalyzes transfer of an amino group from Aspartate to α-Ketoglutarate
• Aspartate à Oxaloacetic Acid (NH3 is removed)
• a-Ketoglutarate à Glutamic Acid (NH3 is transferred to a-KG)
§ Other names
• Serum Glutamic-Oxaloacetic Acid Transaminase (SGOT)
• Aspartate Aminotransferase (ASAT)
§ Tissue sources:
• Widely distributed in human tissues
• Highest concentration: cardiac tissue, liver and skeletal muscle
• Smaller amounts: kidneys, pancreas and erythrocytes
§ Handling
• Hemolysis should be avoided - hemolysis can increase AST activity
• AST activity is stable in serum for 3-4 days at refrigerated temperatures
§ Diagnostic Significance:
• For evaluation of
o Acute Myocardial Infarction – AST begins to rise within 6-8 hours, peak at 24 hrs
after infarction
o Hepatocellular disorders
o Skeletal muscle disorders
§ Karmen Method
• Assay to measure AST
• Coupled enzymatic reaction using Malate Dehydrogenase (MD)
o Indicator of reaction
• Monitors the change of absorbance (340nm) continuously as NADH is oxidized to NAD
• Reagents
o R1: Enzyme Reagent
§ Tris buffer (pH 7.80) – maintains optimum pH
§ L-aspartate – substrate
§ Malate Dehydrogenase – coupled enzyme
§ NADH+H – reducing equivalent, oxidized to NAD+
o R2: Starting Reagent
§ a-Keto Glutarate - substrate

 
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D

o Alanine Transaminase (ALT)


§  Catalyzes transfer of an amino group from Alanine to α-Ketoglutarate
• Alanine à Pyruvic Acid (NH3 is removed)
• a-Ketoglutarate à Glutamic Acid (NH3 is transferred to a-KG)
§ Other names
• Serum Glutamic-Pyruvic Acid Transaminase (SGPT)
• Alanine Aminotransferase (ALAT)
§ Tissue Sources
• Liver (High concentration)
o ALT>AST
o Tends to elevate longer due to longer half-life (serum)
• Cardiac Tissue (Small amounts)
o Normal ALT unless liver damage has occured
o Cardiac tissue contains small amount of ALT activity
§ Handling
• Long half-life: stable for 3-4days at 4ºC
• Unaffected by Hemolysis
§ Diagnostic Significance
• Acute Inflammatory Conditions (Liver)
• Acute Myocardial Infarction (Heart) – differential dx with AST
§ Assay
• Coupled enzymatic reaction using Lactate Dehydrogenase (LD)
o indicator enzyme
• Catalyzes reduction of pyruvate to lactate with simultaneous oxidation of NADH
• Change in absorbance (340nm) is directly proportional to ALT activity
• Reagents
o R1: Enzyme Reagent
§ Tris buffer (pH 7.50) – maintains optimum pH
§ L-alanine – substrate
§ Lactate Dehydrogenase – coupled enzyme
§ NADH+H – reducing equivalent, oxidized to NAD+
o R2: Starting Reagent
§ a-Keto Glutarate - substrate

AST/ALT
• Normal Values

TEMPERATURE 25ºC 30ºC 37ºC


MALE (U/L) 18 25 37
FEMALE (U/L) 15 21 31

• Differential Dx
o AST = marker in both liver and heart
§ Pulmonary Embolism
§ Congestive Heart Disease
§ Hepatocellular disorders
§ Viral Hepatitis
§ Cirrhosis
§ Skeletal muscle disorders
§ Inflammatory conditions
o ALT = marker only in the liver
§ Hepatocellular disorders
§ Acute Inflammatory disorder (liver)